The goal is to define in precise detail at the protein level how the lysyl oxidase-mediated cross-linking mechanisms of the major skeletal tissue collagens differ. Insights and questions from studying heritable disorders make it clear that we do not yet understand the local features of collagen cross-linking chemistry and their underlying control mechanisms well enough to relate form to function. It is also clear that the post- translational quality of collagen can change with age and disease in ways that affect the basic pattern of enzyme-regulated cross-linking. There is growing evidence that such differences between individuals are a risk factor for osteoporosis, osteoarthritis and other degenerative skeletal disorders. The experimental plan targets the biochemistry of collagen cross-linking using advanced mass spectroscopic methods and custom antibodies against cross-linking domains. In parallel, the insights and molecular tools gained from tissue studies will be applied to advance knowledge on existing biomarker assays based on collagen cross-links and develop new biomarkers for monitoring bone and joint disorders non-invasively. The specific aims include: 1) defining the detailed post-translational chemistry of bone collagen including chain usage in the placement and chemical forms of pyridinoline, pyrrole and keto-imine cross-links and differences that occur with age in osteoporosis, osteogenesis imperfecta and other conditions of altered metabolism; 2) compare in similar detail the cross-linking properties of non-mineralized skeletal collagens of cartilages, tendon, ligament andjoint capsule; 3) determine the cross-linking properties of type III collagen as a modifier of the extracellular matrix of cartilage and other non-mineralized skeletal tissues; 4) improving and, developing clinically useful biomarkers from the insights gained on collagen cross-linking mechanisms from the tissue studies. The emphasis in the research design and methods is to apply mass spectrometry and other advanced methods in protein analysis to determine details of tissue collagen structure, then use cell culture to explore candidate control mechanisms responsible for the diversity in tissue-specific cross-linking and the changes that occur in aging and disease.